Membrane proteins (MPs) constitute nearly one third of all human proteins and are known to orchestrate key cellular functions ranging from ion transport, cell-cell attachment, to signaling. Consequently, such proteins are important targets in pharmaceutical drug discovery. However, despite their importance, the direct in vitro study of membrane proteins lacks suitable high throughput screening models, which can be reproducibly fabricated, with controlled lipid composition and where the structural integrity of the protein is preserved upon reconstitution. Bio-appropriate model environments that are simple to construct and are suitable for chip-based applications still remain an important challenge. A key feature of the cell membrane, which is important in membrane protein function is its inherent 2-D fluidity. Lateral diffusion of lipids and membrane proteins within the membrane regulate the distribution of membrane components and affects many processes, such as formation of protein complexes and the dynamic assembly/disassembly lipid disordered and ordered microdomains. Thus, for any artificial bilayer model to be credible, it must exhibit the property of high lateral mobility of lipid and protein constituents.
To this end, while Supported Lipid Bilayers (SLBs) are valuable artificial bilayer models, an inherent major drawback is the interaction of the bilayer with the solid substrate, which dramatically lowers the mobility of the lipids and the incorporated membrane proteins compared with native cell membranes or free liposomes. A number of approaches have been taken to address this issue including, Tethered Bilayer Lipid Membranes (t-BLMs) in order to minimize these interactions by the inclusion of a spacer between the bilayer and the surface. Although t-BLMs were shown to provide better stability to the lipid bilayers, diffusion coefficients of the lipids measured were not significantly improved compared to those measured for SLBs on planar substrates and the same is true for cushioned SLBs. In order to obtain lipid bilayers that are sufficiently separated from the underlying substrates, another approach is to span lipid bilayers across nano- and micro-sized apertures, forming the so-called Black Lipid Membranes (BLM). BLMs however, suffer poor stability due to the retention of organic solvents that are commonly used in their preparation. Moreover, the incorporation and stability of membrane proteins is severely limited owing to their unfavorable mode of preparation and the remnant solvents within the bilayer.
The design of solvent free methods for pore-spanning lipid bilayers is also known in the art. However, most such techniques function in restricted conditions such as the use of certain size of the pores and the vesicles, application of sheer flow and pH, the use of Giant Unilamellar Vesicles (GUVs) or spanning over dry substrates. While each of these methods has shed light on the mechanisms of a variety of pore-spanning lipid membranes, the incorporation and manipulation of membrane proteins within these systems still remains a daunting task.
Typically the best alternatives used are live cell assays. However, these are expensive to implement because of costs associated with cell culture. So an intervening step where the membrane interactions, protein interactions in a biomimetic environment and/or transmembrane transport can be assessed in advance of more complex pre-clinical assessment could offer significant savings.
Porous array structures have been described, e.g. WO2006/104639 describes a porous array where the pores are all sub-micron in dimension. Chemical Communications vol. 47, 2011, ‘Lipid bilayer assembly at a gold nanocavity array’ pp. 12530-12532 Jose et al demonstrates the use of nano-dimensioned pores on arrays with diameters <800 nm. However the limitation of all these structures is that they are sub-micron and to date there have been significant challenges to obtain a bilayer which spans apertures greater than 1 micron.